This invention relates to nucleotide sequences, polypeptides encoded by the nucleotide sequences, and to their use in diagnostic and pharmaceutical applications.
Primary hepatocellular carcinoma (HCC) represents the most common cancer, especially in young men, in many parts of the world (as in China and in much of Asia and Africa) (reviewed in Tiollais, et al., 1985). Its etiology was investigated mostly by epidemiological studies, which revealed that, beyond some minor potential agents such as aflatoxin and sex steroids, hormones, Hepatitis B virus (HBV) chronic infection could account for a large fraction of liver cancers (Beasley and Hwang, 1984).
HBV DNA has been found to be integrated in the genome of most cases of HCCs studied (Edman, et al., 1980; Brechot et al., 1980; Chakraborty, et al., 1980; Chen, et al., 1982). Nonetheless the role of those sequences in liver oncogenesis remains unclear. single HBV integration in a HCC sample in a short liver cell sequence has been reported recently. The sequence was found to be homologous to steroid receptor genes and to the cellular proto-oncogene c-erbA (Dejean, et al., 1986).
Ligand-dependent transcriptional activators, such as steroid or thyroid hormone receptors, have recently been cloned allowing rapid progress in the understanding of their mechanism of action. Nevertheless, there exists a need in the art for the identification of transcripts that may encode for activational elements, such as nuclear surface receptors, that may play a role in hepatocellular carcinoma. Such findings would aid in identifying corresponding transcripts in susceptible individuals. In addition, identification of transcripts could aid in elucidating the mechanisms by which HCC occurs.
Retinoids, a class of compounds including retinol (vitamin A), retinoic acid (RA), and a series of natural and synthetic derivatives, exhibit striking effects on cell proliferation, differentiation, and pattern formation during development (Strickland and Mahdavi, 1978; Breitman et al., 1980; Roberts and Sporn, 1984; Thaller and Eichele, 1987). Until recently, the molecular mechanism by which these compounds exert such potent effects was unknown, although retinoids were thought to modify their target cells through a specific receptor.
Except for the role of retinoids in vision, their mechanism of action is not well understood at the molecular level. Several possible mechanisms have been suggested. One hypothesis proposes that retinoids are needed to serve as the lipid portion of glycolipid intermediates involved in certain, specific glycosylation reactions. Another mechanism, which may account for the various effects of retinoids on target cells, is that they alter genomic expression in such cells. It has been suggested that retinoids may act in a manner analogous to that of the steroid hormones and that the intracellular binding proteins (cellular retinal-binding and retinoic acid-binding protein) play a critical part in facilitating the interaction of retinoids with binding sites in the cell nucleus.
For example, the observation that the RA-induced differentiation of murine F9 embryonal carcinoma cells is accompanied by the activation of specific genes has led to the proposal that RA, like the steroid and thyroid hormones, could exert its transcriptional control by binding to a nuclear receptor (Roberts and Spron, 1984). However, the biochemical characterization of this receptor had been hampered by high affinity RA-binding sites corresponding to the cellular retinoic acid binding protein (CRABP), which is thought to be a cytoplasmic shuttle for RA (Chytil and Ong, 1984).
In any event, retinoids are currently of interest in dermatology. The search for new retinoids has identified a number of compounds with a greatly increased therapeutic index as compared with naturally occurring retinoids. Extensive clinical testing of two of these retinoids, 13-cis-retinoic acid and the aromatic analog etretinate, has lead to their clinical use in dermatology.
In addition, several lines of evidence suggest that important relations exist between retinoids and cancer. A number of major diseases, in addition to cancer, are characterized by excessive proliferation of cells, often with excessive accumulation of extracellular matrix material. These diseases include rheumatoid arthritis, psoriasis, idiopathic pulmonary fibrosis, scleroderma, and cirrhosis of the liver, as well as the disease process atherosclerosis. The possibility exists that retinoids, which can influence cell differentiation and proliferation, may be of therapeutic value in some of these proliferative diseases. There exists a need in the art for reagents and methods for carrying out studies of receptor expression and effector function to determine whether candidate drugs are agonists or antagonists of retinoid activity in biological systems.
There also exists a need in the art for identification of retinoic acid receptors and for sources of retinoic acid receptors in highly purified form. The availability of the purified receptor would make it possible to assay fluids for agonists and antagonists of the receptor.
This invention aids in fulfilling these needs in the art. More particularly, this invention provides a cloned DNA sequence encoding a polypeptide of a newly identified cellular gene, which has been named hap. The DNA sequence has the formula shown in FIG. 2. More particularly, the sequence comprises:
ATGTTTGACTGTATGGATGTTCTGTCAGTGAGTCCTGGGCAAATCCTGGATTTC GTCTTCCTGCATGCTCCAGGAGAAAGCTCTCAAAGCATGCTTCAGTGGATTGACCCAAACCGAATG GCAGCATCGGCACACTGCTCAATCAATTGAAACACAGAGCACCAGCTCTGAGGAACTCGTCCCAAG CCCCCCATCTCCACTTCCTCCCCCTCGAGTGTACATCAAACCCTGCTTCGTCTGCCAGGACAAATCATC AGGGTACCACTATGGGGTCAGCGCCTGTGAGGGATGTAAGGGATGAAGGGCTTTTTCCGCAGAAGTATTCAGAAG AATATGATTTACACTTGTCACCGAGATAAGAACTGTGTTATTAATAAAGTCACCAGGAATCGATGC CAATACTGTCGACTCCAGAAGTGCTTTGAAGTGGGAATGTCCAAAGAATCTGTCAGGAATGACAGG AACAAGAAAAAGAAGGAGACTTCGAAGCAAGAATGCACAGAGAGCTATGAAATGACAGCTGAGTTG GACGATCTCACAGAGAAGATCCGAAAAGCTCACCAGGAAACTTTCCCTTCACTCTGC AAATACACCACGAATTCCAGTGCTGACCATCGAGTCCGACTGGACCTGGGCCTCTGGGACAAATTC AGTGAACTGGCCACCAAGTGCATTATTAAGATCGTGGAGTTTGCTAAACGTCTGCCTGGTTTCACT GGCTTGACCATCGCAGACCAAATTACCCTGCTGAAGGCCGCCTGCCTGGACATCCTGATTCTTAGA ATTTGCACCAGGTATACCCCAGAACAAGACACCATGACTTTCTCAGACGGCCTTACCCTAAATCGA ACTCAGATGCACAATGCTGGATTTGGTCCTCTGACTGACCTTGTGTTCACCTTTGCCAACCAGCTC CTGCCTTTGGAAATGGATGACACAGAAACAGGCCTTCTCAGTGCCATCTGCTTAATCTGTGGAGAC CGCCAGGACCTTGAGGAACCGACAAAAGTAGATAAGCTACAAGAACCATTGCTGGAAGCACTAAAA ATTTATATCAGAAAAAGACGACCCAGCAAGCCTCACATGTTTCCAAAGATCTTAATGAAAATCACA GATCTCCGTAGCATCAGTGCTAAAGGTGCAGAGCGTGTAATTACCTTGAAAATGGAAATTCCTGGA TCAATGCCACCTCTCATTCAAGAAATGATGGAGAATTCTGAAGGACATGAACCCTTGACCCCAAGT TCAAGTGGGAACACAGCAGAGCACAGTCCTAGCATCTCACCCAGCTCAGTGGAAAACAGTGGGGTC AGTCAGTCACCACTCGTGCAATAA.
The invention also covers variants and fragments of the DNA sequence. The DNA sequence is in a purified form.
This invention also provides a probe consisting of a radionuclide bonded to the DNA sequence of the invention.
In addition, this invention provides a hybrid duplex molecule consisting essentially of the DNA sequence of the invention hydrogen bonded to a nucleotide sequence of complementary base sequence, such as DNA or RNA.
Further, this invention provides a polypeptide comprising an amino acid sequence of hap protein, wherein the polypepetide contains the amino acid sequence shown in FIG. 2. More particularly, the amino acid sequence comprises:
MetPheAspCysMetAspValLeuSerValSerProGlyGlnIleLeuAspPheTyrThrAla SerProSerSerCysMetLeuGlnGluLysAlaLeuLysAlaCysPheSerGlyLeuThrGln ThrGluTrpGlnHisArgHisThrAlaGlnserIleGluThrGlnSerThrSerSerGluGlu LeuValProSerProProSerProLeuProProProArgValTyrLysProCysPheValCys GlnAspLysSerSerGlyTyrHisTyrGlyValSerAlaCysGluGlyCysLysGlyPhePhe ArgArgserIleGlnLysAsnMetIleTyrThrCysHisArgAspLysAsnCysvalIleAsn LysvalThrArgAsnArgCysGlnTyrCysArgLeuGlnLysCysPheGluValGlyMetSer LysGluSerValArgAsnAspArgAsnLysLysLysLysGluThrSerLysGlnGluCysThr GluSerTyrGluMetThrAlaGluLeuAspAspLeuThrGluLysIleArgLysAlaHisGln GluThrPheProSerLeuCysGlnLeuGlyLysTyrThrThrAsnserserAlaAspHisArg ValArgLeuAspLeuGlyLeuTrpAspLysPheserGluLeuAlaThrLysCysIleIleLys IleValGluPheAlaLysArgLeuProGlyPheThrGlyLeuThrIleAlaAspGlnIleThr LeuLeuLysAlaAlaCysLeuAspIleLeuIleLeuArgIleCysThrArgTyrThrProGlu GlnAspThrMetThrPheSerAspGlyLeuThrLeuAsnArgThrGlnMetHisAsnAlaGly PheGlyProLeuThrAspLeuValPheThrPheAlaAsnGlnLeuLeuProLeuGluMetAsp AspThrGluThrGlyLeuLeuSerAlaIleCysLeuIleCysGlyAspArgGlnAspLeuGlu GluProThrLysvalAspLysLeuGlnGluProLeuLeuGluAlaLeuLysIleTyrIleArg LysArgArgProSerLysProHisMetPheProLysIleLeuMetLysIleThrAspLeuArg SerIleSerAlaLysGlyAlaGluArgvalIleThrLeuLysMetG.luIleProGlySerMet ProProLeuIleGlnGluMetMetGluAsnSerGluGlyHisGluProLeuThrProSerSer SerGlyAsnThrAlaGluHisSerProSerIleSerProSerSerValGluAsnSerGlyVal SerGlnSerProLeuValGln.
The invention also covers serotypic variants of the polypeptide and fragments of the polypeptide. The polypeptide is free from human serum proteins, virus, viral proteins, human tissue, and human tissue components. Preferably, the polypeptide is free from human, blood-derived protein.
The hap protein (hap for hepatoma) exhibits strong homology with the human retinoic acid receptor (RAR) de The, H., Marchio, A., Tiollais, P. and Dejean, A. Nature 30, 667-670 (1987), Petkovich, M., Brand, N. J., Krust, A. and Chambon, P. Nature 30, 444-450 (1987), a receptor has been recently characterized Petkovich, M., Brand, N. J., Krust, A. and Chambon, P. Nature 30, 444-450 (1987), Giguere, V., Ong, E. S., Segui, P. and Evans, R. M. Nature 30, 624-629 (1987). To test the possibility that the hap protein might also be a retinoid receptor, a chimaeric receptor was created by replacing the putative DNA binding domain of hap with that of the human oestrogen receptor (ER). The resulting hap-ER chimaera was then tested for its ability to trans-activate an oestrogen-responsive reporter gene, (vit-tk-CAT) in the presence of possible receptor ligands. It was discovered that retinoic acid (RA) at physiological concentrations is effective in inducing the expression of this reporter gene by the hap-ER chimaeric receptor. See Nature, 332:850-53 (1988). This demonstrates the existence of two human retinoic acid receptors designated RAR-xcex1 and RAR-xcex2.
More particularly, it has been discovered that the hap protein is a second retinoic acid receptor. Thus, the expression xe2x80x9chap proteinxe2x80x9d is used interchangeably herein with the abbreviation xe2x80x9cRAR-xcex2xe2x80x9d for the second human retinoic acid receptor.
Also, this invention provides a process for selecting a nucleotide sequence coding for hap protein or a portion thereof from a group of nucleotide sequences comprising the step of * determining which of the nucleotide sequences hybridizes to a DNA sequence of the invention. The nucleotide sequence can be a DNA sequence or an RNA sequence. The process can include the step of detecting a label on the nucleotide sequence.
Still further, this invention provides a recombinant vector comprising lambda-NM1149 having an EcoRI restriction endonuclease site into which has been inserted the DNA sequence of the invention. The invention also provides plasmid pCOD20, which comprises the DNA sequence of the invention.
This invention provides an E. coli bacterial culture in a purified form, wherein the culture comprises E. coli cells containing DNA, wherein a portion of the DNA comprises the DNA sequence of the invention. Preferably, the E. coli is stain TG-1.
In addition, this invention provides a method of using the purified retinoic acid receptor of the invention for assaying a medium, such as a fluid, for the presence of an agonist or antagon-ist of the receptor. In general, the method comprises providing a known concentration of a proteinaceorus receptor of the invention, incubating the receptor with a ligand of the receptor and a suspected agonist or antagonist under conditions sufficient to form a ligand-receptor complex, and assaying for ligand-receptor complex or for free ligand or for non-complex receptor. The assay can be conveniently carried out using labelled reagents as more fully described hereinafter, and conventional techniques based on nucleic acid hybridization, immunochemistry, and chromotography, such as TLC, HPLC, and affinity chromatography.
In another method of the invention, a medium is assayed for stimulation of transcription of the RAR-xcex2 gene or translation of the gene by an agonist or antagonist. For example, xcex2-receptor binding retinoids can be screened in this manner.